CN102676601B - Preparation method of lovastatin - Google Patents
Preparation method of lovastatin Download PDFInfo
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- CN102676601B CN102676601B CN201110065427.6A CN201110065427A CN102676601B CN 102676601 B CN102676601 B CN 102676601B CN 201110065427 A CN201110065427 A CN 201110065427A CN 102676601 B CN102676601 B CN 102676601B
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- lovastatin
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Abstract
The invention discloses a preparation method of lovastatin. The preparation method comprises steps of fermenting aspergillus strains in a fermentation medium to obtain the lovastatin. The preparation method is characterized in that sterilized biotin solution and/or zinc ion solution are/is added in the fermentation medium. The preparation method effectively improves yield of the lovastatin and reduces production cost.
Description
Technical field
The present invention relates to a kind of preparation method of lovastatin, particularly relate to a kind of method being prepared lovastatin by fermentation method.
Background technology
Lovastatin (lovastatin, also MonacolinK is claimed, monacolin K) be that one derives from microorganism HMG-CoA (hydroxy-methyl-glutaryl coenzyme A) reductase inhibitor, it is the medicine being used for the treatment of high-cholesterol disease, it can stop arteriosclerosis to develop, reduce the danger of the morbidities such as myocardial infarction, therefore medically significant.The problems such as in current production, ubiquity output is on the low side, production cost is higher.
At present, the microorganism that production lovastatin adopts mainly contains three major types: Penicillium notatum, red mould and terreus, wherein terreus is bacterial classification the most frequently used in industrial production.Domestic research it mainly concentrates on pellet form change in Study on extraction and fermenting carbon source, nitrogenous source optimization aspect and fermenting process.The research of its fermenting process is mainly concentrated on to the research of lovastatin biosynthetic pathway and precursor thereof abroad.There is not been reported on the impact of aspergillus terreus (Aspergillus terreus) fermentation generation lovastatin output for metal ion and vitamin H.
Summary of the invention
For solving the problem, the object of this invention is to provide a kind of preparation method of lovastatin, thus effectively improving output, reduce production cost.
For achieving the above object, the preparation method of a kind of lovastatin provided by the present invention, comprise the step fermenting to obtain lovastatin in the fermentation medium to terreus bacterial classification, it is characterized in that, in fermention medium, add sterilized biotin solution and/or zine ion solution during the fermentation.
According to a specific embodiment of the present invention, in described preparation method, in fermention medium, add sterilized biotin solution or zine ion solution during the fermentation, making biotin concentration in fermention medium be 0.00002 ‰-0.0002 ‰ (w/w) or zinc ion concentration is 0.5mM-9mM; Preferably in fermention medium, biotin concentration is 0.00002 ‰-0.00012 ‰ (w/w) or zinc ion concentration is 1mM-7mM.
According to of the present invention one preferred embodiment, in described preparation method, in fermention medium, add sterilized biotin solution and zine ion solution during the fermentation, making biotin concentration in fermention medium be 0.00002-0.0002 ‰ (w/w) and zinc ion concentration is 0.5mM---9mM; Preferably in fermention medium, biotin concentration is 0.00006-0.00012 ‰ (w/w) and zinc ion concentration is 1mM-7mM.
Preferably, in the 0-36h process after fermentation starts, in fermention medium, add sterilized biotin solution and/or zine ion solution, and then proceed fermentation.Applicant finds, add sterilized biotin solution and/or zine ion solution at the fermentation initial stage, effect is better.Condition those skilled in the art of other fermentation can select according to common fermentation processes.Such as, in fermentation medicine bottle, fermentation 180-230 hour can be carried out with 200r/min in the rotary shaker of 26 ± 1 DEG C.
In the preparation method of lovastatin of the present invention, before being usually also included in fermentation, bacterial classification is carried out in seed culture medium the step of cultivating, and then the seed liquor obtained after cultivation is inoculated in fermention medium, ferment.Preferably, the condition of carrying out cultivating in seed culture medium for cultivate 90-96h at temperature 26 ± 1 DEG C.Preferably, the seed liquor obtained after cultivation is inoculated in fermention medium with the inoculum size of 5-15%.
In the preparation method of lovastatin of the present invention, usually also comprise the step of after fermentation fermented liquid being carried out to separating-purifying.Such as fermented liquid filtered, extract, crystallization, drying, to obtain lovastatin finished product.
According to a specific embodiment of the present invention, can first by strain inoculation in seed culture medium, 90-96h is cultivated at temperature 26 ± 1 DEG C, then by 10% inoculum size, the seed liquor obtained after cultivation is inoculated in fermention medium, 200r/min, 26 ± 1 DEG C of rotary shaker ferment, in fermentation 0-36h process, the sterilized biotin solution of independent interpolation or zine ion solution, making biotin concentration in fermentation flask substratum be 0.00002 ‰-0.00012 ‰ (w/w) or zinc ion concentration is 0.5mM---9mM, or compound adds sterilized biotin solution and zine ion solution, making biotin concentration in fermentation flask substratum be 0.00006-0.00012 ‰ (w/w) and zinc ion concentration is 0.5mM---9mM.Cultivate 216h; Processed routinely by gained fermented liquid, filtration, extraction, crystallization, drying, obtain lovastatin finished product.
In the present invention, fermention medium can conveniently compound method be prepared.Such as, fermention medium can be (by weight percentage): 20% glucose, 4% dregs of beans, 0.5% malt extract, 1% peptone, 0.1%NaCl, 0.05%MgSO
4, 0.05%KH
2pO
4, 0.1%NaNO
3, surplus is water, pH6.8.
In the biosynthetic process of lovastatin, first be acetyl-CoA and malonyl coenzyme A through polyketide synthases synthesizing dihydro not that crin L of LovB, LovC genes encoding, under the effect of the P450 oxygenase of LovA genes encoding, be then oxidized to intermediate not that crin J.Then catalysis acetic acid and propanedioic acid units condense are 2-methylbutyryl side chain to the diketone synthetic enzyme of simultaneously LovF genes encoding; and under the effect of the acyltransferase (AT) of LOvD genes encoding, the C-8 position hydroxyl being transferred to not that crin J molecule becomes final product lovastatin.Experiment finds configuration metal ions Zn
2+affect the activity of relevant enzyme in the biosynthetic process of lovastatin.In the present invention, zine ion can being added by adding zinc sulfate in fermention medium, also can realize by adding zinc chloride etc.
Present inventor improves discovery surprised in the process of lovastatin output in research, in the fermention medium of terreus, add configuration metal ions Zn
2+and vitamin H, the output of lovastatin can be improved.Use method provided by the invention, by adding configuration metal ions Zn in lovastatin fermention medium
2+and vitamin H, be no matter independent add configuration metal ions Zn
2+or vitamin H, still add the configuration metal ions Zn of compound
2+and vitamin H, all can significantly improve the biosynthesizing of lovastatin.And, when adding configuration metal ions Zn in fermention medium
2+during with vitamin H, configuration metal ions Zn
2+and can synergy be played between vitamin H, significantly improve the output of lovastatin.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only for illustration of the present invention but not for limiting scope of the present invention.
Unless otherwise indicated, the % used in following examples is weight ratio.
In an embodiment of the present invention, inclined-plane (i.e. bacterial classification) is inoculated in shake-flask seed substratum, 26 ± 0.5 DEG C of shaking speed 200 revs/min, cycle 90-96 hour; By in 10% inoculum size access Medium of shaking flask fermentation, fermention medium compound method preparation routinely, the formula of fermention medium is: 20% glucose, 4% dregs of beans, 0.5% malt extract, 1% peptone, 0.1%NaCl, 0.05%MgSO
4, 0.05%KH
2pO
4, 0.1%NaNO
3, surplus is water, pH6.8, leavening temperature is 26 ± 1 DEG C.
In an embodiment of the present invention, the assay of lovastatin adopts HPLC, detects according to high performance liquid chromatography (Chinese Pharmacopoeia 2000 editions two annex VD).
embodiment 1add configuration metal ions Zn
2+on the impact of lovastatin output
The fermention medium of preparation lovastatin, and ZnSO is added respectively in lovastatin fermention medium
4, make Zn
2+concentrations by weight in corresponding substratum is respectively 1mM, 3mM, 5mM, 7mM, meanwhile, not add ZnSO
4the original fermention medium of lovastatin substratum in contrast, access aspergillus terreus (Aspergillus terreus) fermentation culture after 216 hours, the content of sampling and measuring lovastatin.Result is as shown in table 1.
Table 1, interpolation ZnSO
4on the impact of lovastatin output
| ZnSO 4Dosage ‰ | 0 | 1mM | 3mM | 5mM | 7mM |
| Relative content | 100 | 108 | 116 | 115 | 109 |
According to the result of table 1.Add different concns configuration metal ions Zn SO in the medium
4after, the output of lovastatin there occurs change in various degree, wherein, adds the Zn of 1--7mM
2+after, the output of lovastatin obtains and significantly improves.
embodiment 2add vitamin H to the impact of lovastatin output
The fermention medium of preparation lovastatin, and vitamin H is added respectively in lovastatin fermention medium, the concentrations by weight of vitamin H in corresponding substratum is made to be respectively 0.00002 ‰, 0.00006 ‰, 0.0001 ‰, 0.00012 ‰, simultaneously, with the original fermention medium of the lovastatin not adding vitamin H substratum in contrast, access aspergillus terreus (Aspergillus terreus) fermentation culture after 216 hours, the content of sampling and measuring lovastatin.Result is as shown in table 2.
Table 2, interpolation vitamin H are on the impact of lovastatin output
| Vitamin H dosage ‰ | 0 | 0.00002 | 0.00006 | 0.0001 | 0.00012 |
| Relative content | 100 | 105 | 109 | 108 | 107 |
According to the result of table 2.After adding different concns vitamin H in the medium, the output of lovastatin there occurs change in various degree, and wherein, after adding the vitamin H of 0.00002 ‰-0.00012 ‰, the output of lovastatin obtains and significantly improves.
embodiment 3compound adds configuration metal ions Zn SO
4with the impact of vitamin H on lovastatin output
3.1ZnSO
4with the mutual test of vitamin H
According to the result of embodiment 1, embodiment 2, select ZnSO
4carry out compound with vitamin H and add experiment, and respectively centered by their respective the suitableeest interpolation concentration, according to the cultural method of embodiment 1, make ZnSO
4with the interaction of vitamin H, meanwhile, not add ZnSO
4with the original fermention medium of the lovastatin of vitamin H substratum in contrast, cultivate and detect the output of the lovastatin in final cultures, result is as table 3.
Table 3, ZnSO
4with the hydraulic test table that vitamin H combination is added
| ZnSO 4 | Vitamin H ‰ | Relative content |
| 1mM | 0.00006 | 121 |
| 3mM | 0.00006 | 118 |
| 5mM | 0.00012 | 120 |
| 7mM | 0.00012 | 116 |
| 0 | 0 | 100 |
According to the result of table 3, compound adds ZnSO
4the output of lovastatin can be improved with vitamin H, and compare single interpolation ZnSO
4or vitamin H, compound adds ZnSO
4better with the effect of vitamin H, ZnSO
4and between vitamin H, there is synergy, and its concentration range is respectively: 1-7mM, and 0.00006 ‰-0.00012 ‰.
3.2 compounds add configuration metal ions Zn SO
4the time is added on the impact of lovastatin output with vitamin H
The fermention medium of preparation lovastatin, access aspergillus terreus (Aspergillus terreus) fermentation culture, respectively at 0,12,36,60,84,108 hour, adds ZnSO respectively in the fermention medium of lovastatin
4, vitamin H, make Zn
2+, vitamin H concentrations by weight reach 5mM, 0.00004 ‰ respectively, meanwhile, not add ZnSO
4, vitamin H the original fermention medium of lovastatin substratum in contrast, cultivate and detect the content of the lovastatin in final cultures, result is as shown in table 4.
Table 4, compound add ZnSO
4the time is added on the impact of lovastatin output with vitamin H
| Interpolation time (h) | 0 | 12 | 36 | 60 | 84 | 108 | Contrast |
| Relative content | 120 | 118 | 117 | 109 | 110 | 108 | 100 |
According to the result of table 4, in fermention medium, add Zn at earlier fermentation
2+(5mM), vitamin H (0.00004 ‰) can significantly improve the biosynthesizing of lovastatin, and the preferred interpolation time is 0-36 hour.
In sum, use method provided by the invention, in the fermention medium of lovastatin, add ZnSO
4and vitamin H, no matter be add ZnSO separately
4or vitamin H, still add compound ZnSO
4and vitamin H, fermentation culture lovastatin, all can significantly improve the biosynthesizing of lovastatin.
Although described out preferred embodiment of the present invention herein, the scope that the many changes clearly can carried out to those skilled in the art and amendment can not exceed from broad aspect of the present invention.Therefore, appended claim covers all changes within true spirit of the present invention and scope and amendment to attempt.
Claims (7)
1. a preparation method for lovastatin, comprises the step fermenting to obtain lovastatin in the fermentation medium to terreus bacterial classification, it is characterized in that, adds sterilized biotin solution and zine ion solution during the fermentation in fermention medium,
Wherein, when adding sterilized biotin solution and zine ion solution during the fermentation in fermention medium, making biotin concentration in fermention medium be 0.00006 ‰ (w/w) and zinc ion concentration is 1mM,
Wherein, when fermenting beginning, in fermention medium, add sterilized biotin solution and zine ion solution, and then proceed fermentation.
2. preparation method according to claim 1, is characterized in that, fermentation condition is at 26 ± 1 DEG C of bottom fermentation 180-230 hour.
3. preparation method according to claim 1, is characterized in that, before being also included in fermentation, bacterial classification is carried out in seed culture medium the step of cultivating, and then is inoculated in fermention medium by the seed liquor through cultivating, and ferments.
4., according to described preparation method arbitrary in claim 1-3, it is characterized in that, bacterial classification inoculum size is in the fermentation medium 5-15%.
5., according to described preparation method arbitrary in claim 1-3, it is characterized in that the preparation method of described lovastatin also comprises the step of after fermentation fermented liquid being carried out to separating-purifying.
6., according to described preparation method arbitrary in claim 1-3, it is characterized in that, fermention medium is by weight percentage: 20% glucose, 4% dregs of beans, 0.5% malt extract, 1% peptone, 0.1%NaCl, 0.05%MgSO
4, 0.05%KH
2pO
4, 0.1%NaNO
3, surplus is water, pH6.8.
7. according to described preparation method arbitrary in claim 1-3, it is characterized in that, in fermention medium, add zine ion realized by interpolation zinc sulfate or zinc chloride.
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| CN105695527A (en) * | 2016-04-27 | 2016-06-22 | 山东鲁抗医药股份有限公司 | Culture medium for lovastatin fermentation and supplementing method for fermentation process |
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Non-Patent Citations (3)
| Title |
|---|
| J.L. Casas López, J.A. Sánchez Pérez, J.M. Fernández Sevilla.Production of lovastatin by Aspergillus terreus: effects of the C:N ratio and the principal nutrients on growth and metabolite production.《Enzyme and Microbial Technology》.2003,第33卷(第2-3期),2 材料与方法 2.1. * |
| 洛伐他汀发酵培养基配方优化及15L罐放大;吴波, 陈长华, 杨琳;《中国抗生素杂志》;20070731;第32卷(第7期);正文第2段，1 材料与方法 1.1 菌种和培养基 * |
| 贾志华,张小里,曹学君,秦宝福,刘建党.复合氮源对土曲霉洛伐他汀生物合成的影响.《西北农林科技大学学报》.2010,第38卷(第10期),摘要,正文左栏第3段. * |
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Owner name: TOPFOND PHARMACEUTICAL CO., LTD. Free format text: FORMER OWNER: TIANFANG PHARMACEUTICAL CO., LTD., HENAN Effective date: 20150716 |
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Effective date of registration: 20150716 Address after: Zhumadian City, Henan province 463000 Guangming Road Yicheng District No. 2 Patentee after: TIANFANG PHARMACEUTICAL CO., LTD. Address before: 463000 No. 2 Guangming Road, Henan, Zhumadian Patentee before: Tianfang Pharmaceutical Co., Ltd., Henan |